Sepsis is a major threat to human health in the US, affecting ~750,000 Americans per year, with an associated mortality rate >28%. Many septic patients develop profound immunosuppression manifested by decreased production of pro-inflammatory cytokines and chemokines. This is highly reminiscent of endotoxin tolerance, a state of re-programming of TLR4 responses after a prior exposure to LPS. Endotoxin tolerance can be used as a model to delineate mechanisms that underlie altered monocyte responses in patients with sepsis. During the period of funding, we have identified new hallmarks of endotoxin tolerance. These include: (i) deficient tyrosine phosphorylation of TLR4 and adapter protein Mal; (ii) suppressed LPS-induced signalosome assembly amongst TLR4, adapter protein TRIF and kinase TBK1; (iii) deficient activation of TBK1 and transcription factor IRF-3; and (iv) increased expression of negative regulators Tollip, IRAK-M, SHIP-1, SOCS-1, SARM, and SIKE. Our preliminary data in THP1 cells and human monocytes show decreased LPS- induced c-Src and Lyn phosphorylation and Lyn-TLR4 interactions; inhibited activation of kinases IRAK4 and TAK-1; impaired K63-linked polyubiquitination of IRAK1 and TRAF-6; suppressed interactions of ubiquitinated IRAK1 with TRAF6 and IKK3; and increased expression of A20, a key deubiquitinating enzyme. Furthermore, we found reduced LPS tolerance induction upon inhibition of protein tyrosine phosphatases or by A20 gene knockdown, indicating a crucial role for altered tyrosine phosphorylation and K63-linked polyubiquitination in tolerance. Based on these data, we hypothesize that LPS tolerance alters post-translational modifications of TLR4, Mal and IRAK kinases and increases levels of negative regulators of TLR signaling, leading to reprogramming of TLR responses through changes in proximal signalosome compositions. This hypothesis will be tested in the following Specific Aims: 1. Identify kinases and phosphatases involved in phosphorylation of TLR4 and Mal and determine the impact of LPS tolerance on their expression and activities; 2. Define mechanisms by which LPS tolerance alters signalosome assembly and activation of proximal adapter-kinase modules; and 3. Elucidate molecular basis of interference in TLR4 signaling by negative regulatory molecules associated with LPS tolerance. These studies will determine new mechanisms responsible for tolerance and identify key intermediates affected. They will lay the groundwork for our future research in animal models of sepsis in vivo and translational studies in septic patients, with the goal of facilitating development of new therapeutic strategies to improve treatments for septic patients. These advances would be of key importance for improving public health in septic patients in the U.S.